In the floriculture industry, it is important to develop new and different varieties of flowering plants. In particular, regarding flower colour, which is one of the most important characteristics of flowering plants, classical breeding techniques that relies on crossing have been used to develop new varieties exhibiting various colours. However, since genetic resources are very limited among a particular plant species in which crossing can be carried out, it is rare for a single plant species to have a full spectrum of colour varieties.
Flower colour is predominantly due to a class of compounds, generally called anthocyanins, which belong to flavonoids. It has been known that there are various anthocyanins in plants, and the molecular structures of many of these compounds have already been determined. The colour of an anthocyanin is determined mainly by its structures (Harborne (1986) The Flavonoids, p. 565). Research has been conducted on enzymes, and genes encoding these enzymes, involved in biosynthesis of anthocyanins. There are some examples in which structure of anthocyanins were modified to alter flower colours by molecular biological techniques, and introduction of genes in plants (Holton et al. (1995) Plant Cell, 7, p. 1071; Tanaka et al. (1998) Plant Cell Physiol. 39. p. 1119).
The biochemical pathway for biosynthesis of anthocyanins up to anthocyanidin 3-glucosides is common in most flowering plants (Holton et al. (1995) Plant Cell, 7, p. 1071). Thereafter, anthocyanidin 3-glucosides present in plants are subjected to diverse modifications in a species- or varieties-specific manner. The diversity of this modification is one of the reason for the diversity of flower colours.
It is known that, although anthocyanins are unstable compounds in neutral solution, their stability is improved by modification with a glycosyl or an acyl group (Forkmann (1991) Plant Breeding, 106, p. 1). It is also known that they become bluer when an aromatic acyl group is added (Forkmann (1991) Plant Breeding, 106, p 1). It should be noted that the acyl group is not bound directly to the skeleton of the anthocyanidins, but indirectly via a glycosyl group that is bound to the anthocyanidin. Thus, in order for the stabilization and blue colour to be achieved by addition of the acyl group, it is necessary as a prerequisite that a glycosyl group have been added to the anthocyanidin.
In representative species of flowering plants exhibiting blue flower colour such as gentian, cineraria, and butterfly pea, their anthocyanins are modified with a glucose at the 3′-position of the B ring and the glucose is further modified with an aromatic acyl group (Yoshida et al. (1992) Tetrahedron, 48, p. 4313; Goto et al. (1984) Tetrahedron Letters, 25, p. 6021; Goto et al. (1991) Angrew. Chem. Int. Ed. Engl. 30, p. 17; respectively). Although it has been shown from a study using the main pigment of a gentian that the acyl group at the 3′-position of the B ring contributes to the stabilization and bluer colour of anthocyanidin (Yoshida et al. (2000) Phytochemistry 54, p. 85), it is prerequisite on the condition that a glycosyl group have been added to the 3′-position.
Several studies have been reported on glycosylation of flavonoids. For example, nucleic acids encoding enzymes that ctalyze a reaction to transfer a glucose molecule to a hydroxyl group at the 3-position of anthocyanidins have been cloned from snapdragon, gentian, perilla, barley, and corn (e.g. Tanaka et al. (1996) Plant Cell Physiol., 37, p. 711). Also, nucleic acids encoding enzymes that catalyze a reaction to transfer glactose to a hydroxyl group at the 3-position of anthocyanidins have been cloned from vigna mungo, and petunia (Mato et al. (1998) Plant Cell Physiol. 39, p. 1145; Miller et al. (1999) J. Biol. Chem. 273, p. 34011).
Nucleic acids encoding enzymes that catalyze a reaction to transfer glucose to a hydroxyl group at 5-position of anthocyanins have been cloned from perilla, verbena, and torenia (WO99/05287). A nucleic acid encoding enzymes that catalyze a reaction to transfer rhamnose to anthocyanidin 3-glucoside has been cloned from petunia (Brugliera et al. (1994) Plant J. 5, p. 81).
Also, a nucleic acid encoding enzymes that catalyze a reaction to transfer glucose to a hydroxyl group at the 7-position of flavonoids has been cloned from Scutellaria baicalensis, and it has been reported that a protein obtained by expressing this gene in Escherichia coli also catalyzes a reaction to transfer glucose to a hydroxyl group at the 7-position of flavonoids (Suzuki et al. (2000) Plant 210, p. 1006). A nucleic acid encoding enzymes that catalyze a reaction to transfer glucose to a hydroxyl group at the 5-position of betanidine has been cloned, and a protein obtained by expressing this gene in Escherichia coli catalyzes a reaction to transfer glucose to a hydroxyl group at the 4′- and 7-positions of flavonoids (Vogt et al. (1999) Plant J. 19:509–519).
However, there has been no report on nucleic acid encoding enzymes that catalyze a reaction to transfer a glucose to the 3′-position of anthocyanins, and activity of such a enzyme has not been measured. No such enzyme has been purified, nor has any nucleic acid encoding such an enzyme ever been cloned. Glycosyltransferase may catalyze reactions of transferring glucose to plural hydroxy groups, as described above. However, in order to accumulate a target anthocyanin in a plant, it is necessary to use an enzyme exhibiting high substrate-specificity.